Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage

Abstract

Introduction: Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration.

Methods: sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness.

Results: All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation.

Conclusions: Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced subchondral sclerosis, synovial macrophage activation, and osteophyte formation.

Figure 1

Hypothetical model that shows in what manner changes of cartilage, subchondral bone, and synovial macrophages all contribute to osteoarthritis development. (A) Schematically depicted healthy joint with chondrocytes in cartilage extracellular matrix, bone and inactive osteoclasts, and resting synovial macrophages. (B) Chondrocytes with a pathological strain produce cytokines and growth factors that diffuse toward the underlying bone marrow and synovium. There these products stimulate osteoclastogenesis and can activate macrophages. (C) Progressive phase of OA. Chondrocytes become hypertrophic and produce less sulfated-glycosaminoglycans (sGAGs) to sustain the cartilage, making the ECM more susceptible to compressive forces. Osteoclasts start tunneling through the subchondral bone, which compromises plate stability, and changing its supportive function for the overlying cartilage. Activated synovial macrophages produce growth factors of their own that promote synovial fibrosis, osteophyte formation, and may stimulate ECM degradation. (D) Eventually, cartilage is severely sGAG depleted and becomes structurally deprived. Activated macrophages stimulate fibrotic remodeling of the synovium and induce osteophyte growth. Osteoclast activity extends into the calcified cartilage, up to the border with the deep zone of the cartilage. Through subchondral pores, vascular ingrowth occurs into the cartilage. Later, osteoblasts infiltrate and start to deposit bone that results in end-stage sclerosis.

Figure 2

Experiment design indicating analytic time points and methods for each experimental group. Forty 16-week-old male Wistar rats received three intraarticular papain injections (P.I.) and divided over two different groups: a sedentary group (n = 20) and a running group (n = 20). All running rats were subjected to a 6-week moderate running protocol, earlier reported to protect against OA [17]. During the experiment, three μCT scans were made to measure longitudinal subchondral bone changes. At 6 and 12 weeks, a full analysis sequence was done in 10 animals per group (n^†), consisting of: determination of activated macrophages by using SPECT/CT in vivo; and cartilage analysis with equilibrium partitioning of an ionic contrast agent by using (EPIC-)μCT and histology ex vivo.

Figure 3

Cartilage quality and quantity was determined from samples of sedentary (round boxes) and running (square boxes) rats with equilibrium partitioning of a ionic contrast agent by using (EPIC-)μCT (A-D). The amount of sulfated-glycosaminoglycans (sGAGs) (arbitrary gray values; A, B) and cartilage thickness (μm; C, D) were measured of medial (A,C) and lateral (B,D) cartilage compartments of the tibial plateau harvested from control (blank boxes) and sGAG depleted joints (gray boxes). Attenuation values from EPIC-μCT scans are inversely related to the sGAG content, meaning that a high attenuation corresponds to low sGAG content. Coronal images from representative EPIC-μCT scans of the tibial plateau show the amount of cartilage (erosions indicated with ▼ and dashed lines) and sGAG content (displayed in color) (E). * < 0.05, **P < 0.01, ***P < 0.001; error bars indicate 95% confidence intervals.

Figure 4

Histology of safranin-O-stained sagittal sections of medial and lateral tibia plateau cartilage after 6 weeks and 12 weeks of follow-up. Both in sedentary and in running animals, cartilage of papain-injected joints was severely sulfated-glycosaminoglycan depleted at 6 weeks and 12 weeks. But in running animals, the extracellular matrix showed clear signs of erosion in the medial compartment and even denudation of bone in the lateral compartment.

Figure 5

Subchondral bone changes analyzed with longitudinal in vivo μCT in control (white circles) and papain injected (gray squares) knee joints. Data points were nudged from analyzed time points 0, 6, and 12 weeks for clearer representation of results. Subchondral plate thickness (Sb. Pl. Th.; A, C) and porosity (Sb. Pl. Por.; B, D) were measured in the medial (A,B) and lateral (C,D) compartments of the tibial epiphysis. Changes in trabecular thickness (Tb. Th.; E) and trabecular bone volume fraction (BV/TV; F) were measured in tibial epiphysis bone marrow. (G) Representative sagittal images from binary μCT scans to show most prominent subchondral bone changes, pore development in medial subchondral bone of papain-injected animals (▼), and development of subchondral sclerosis (dashed line and *) in lateral subchondral bone of papain-injected and running animals. Three-dimensional top views of the tibial plateau at different time points (H) show subchondral pore (red color) development in papain animals and papain-plus-running animals. *P < 0.05; **P < 0.01; ***P < 0.001; error bars indicate 95% confidence intervals.

Figure 6

Macrophage activation determined in sedentary (round boxes) and running (square boxes) rats by injection of [111In]-DOTA-Bz-folate by using SPECT/CT. (A) Quantitative outcome of measured radioactivity in the control (blank boxes) and papain-injected (gray boxes) knee joints normalized to the size of the cylindrical region of interest (kBq/mm³). Absolute differences per animal were calculated (kBq/mm³) to reduce interindividual variation (black boxes). A high radioactivity is related to more macrophage activation. (B) Ectopic bone formation (mm³) as a measure for osteophyte development was quantified on longitudinal bone μCT scans. (C) Sagittal SPECT/CT images of knee joints from representative animals. CT images shown in black and white were used for anatomic reference; the SPECT images are shown in color. Transaxial images from patellar bone extracted from binary μCT images show ectopic bone formation (red color). *P < 0.05; **P < 0.01; ***P < 0.001; error bars indicate 95% confidence intervals.